A wide spectrum of degenerative diseases have now been associated with defects in the mitochondrion. Because of the mitochondrion's central function in cellular metabolism and its assembled from approximately 1000 nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) genes, mitochondrial diseases are phenotypically and genetically complex. A prime example of this complexity is the mitochondrial respiratory complex I which is assembled from seven mtDNA genes and 36 nDNA genes. Patients with complex I defects can have phenotypes ranging from midlife-onset optic atrophy to lethal childhood Leigh disease. Inheritance patterns can be maternal or mendelian, but many others appear aqs either spontaneous" or complex. We hypothesize that much of this genetic complexity is due to the faulty interaction between nDNA and mtDNA genes. To test this hypothesis, we have perfected a series of high-through-put genomic analysis methods for the mtDNA and nDNA complex I genes, including the development of our novel "Mitochip" DNA microarrays for analyzing mitochondrial gene expression. We are now applying these methods to our large collection of complex I disease patients and have already identified several novel mtDNA mutations as well as dominant and recessive DNA mutations. Completion of these studies should give us a clearer view of the relative importance of single versus multiple-gene defects for complex I diseases. To further investigate the genetics and pathophysiology of mitochondrial disease, we have also developed procedures for introducing mitochondrial gene mutations into the mouse mitochondrial and nuclear genome. We now propose to use these procedures to generate mice harboring various mtDNA and nDNA complex I mutations. These mtDNA and nDNA mutants will then be mixed by either standard mating or by nuclear-transplantation of mutant nuclei into enucleated oocytes harboring various mutant mtDNAs. Comparison of the phenotypes and biochemical and molecular defects of these nDNA + mtDNA mutant mice with their parental strains should clarify the nature and importance of aberrant nuclear-cytoplasmic in human mitochondrial disease.